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Fruit cells are living irregular three‐dimensional (3D) transparent objects which makes them challenging to determine their real 3D size and shape through only two‐dimensional (2D) images using the existing biological microscope. This study deals with a newly self‐developed biological microscope including a microscope imaging system, a light source system, a stage and a support base for the 3D size characterization of fruit single cells. The main design concept is based on two optical path systems set up at the front (x‐axis) and bottom (z‐axis) directions of a transparent chamber containing single cells that allow the front view and bottom view of the single cell to be observed. Performance indicators such as mass, size, observation range, objective magnification, total magnification, focal range, focal accuracy, and resolution of the developed biological microscope were estimated. Finally, the 3D geometry size of single tomato cells was measured by the new biological microscope to demonstrate the relative ease at which accurate real 3D geometry information of single fruit cells could be obtained, which echoes its scientific value. [ABSTRACT FROM AUTHOR]